The proposed project will develop cutting edge analytical chemistry methodology for the detection of endogenous mu-opioid receptor peptide ligands as candidate biomarkers in heroin addiction. The application of mass spectrometry to the quantification of peptide hormones in plasma has been a long sought goal. The immense molecular complexity of plasma, with a vast number of distinct molecular entities present over a wide range of concentrations, however, has proven to be a challenging obstacle. Our recent success, identifying endogenous vasopressin in rat plasma (Reed et al., Endocrinology, 2009, 150, 2934-2939), demonstrated for the first time the use of targeted mass spectrometry with successive stages of fragmentation (ms3) for the detection of endogenous peptides present in the picomolar concentration range. Our goal is to develop and optimize innovative analytical chemistry methodology coupling capillary chromatography with ion trap mass spectrometry capable of targeted ms3 detection with innovative stable isotope labeled peptide standards for the parallel quantification of multiple peptide hormones, initially focusing on putative mu-opioid receptor peptide ligands (-endorphin, Met-enkephalin, Met-enkephalin-Arg-Phe, Met-enkephalin-Arg-Gly-Leu, and Leu- enkephalin). The potential of parallel immunoaffinity enrichment of -endorphin/enkephalins from plasma for increasing the sensitivity of targeted ms3 detection will be systematically determined. The optimized sample preparation and analytical methodology will then be used to analyze plasma samples from rat, establishing intra- and inter-animal variability of the endogenous opioid peptide ligand levels as well as reponsivity to the mu-opioid receptor antagonist naloxone. These studies form the basis of the R21 portion of this study, designed to A - evaluate and extend the potential of targeted ms3 mass spectrometry to the challenge of unambiguously quantifying multiple peptide hormones in parallel in plasma samples, and B - demonstrate the feasibility of alterations of the endogenous mu-opioid receptor peptide ligands in response to endocrine challenge. In the R33 proposal, we aim to apply the novel quantification techniques to healthy human subjects, to determine the baseline levels of these peptide hormones in human plasma, as well as potential differences with respect to genotypic variants of the mu-opioid receptor, namely the A118G polymorphism. We hypothesize that we will observe alterations in the plasma levels of -endorphin, as well as potentially of the pro-enkephalin derived peptides, following stimulation of the HPA axis following endocrine challenge, including innovative direct comparisons of vasopressin and corticotrophin releasing hormone. We will then apply these methods to a translational study of the major pharmacotherapy for heroin addiction, methadone maintenance treatment in a novel rat model, to determine alterations in peptide hormone biomarker levels, which will have impact both for increasing our understanding of the endocrinological basis of heroin addiction and potentially improving patient treatment.